Exotic plantations differ in “nursing” an understory invader: A probe into invasional meltdown

Abstract Forest plantations most likely promote exotic plant invasion. Using an in situ monitoring method, this study investigated the traits correlated with growth and reproduction of an understory invader, Phytolacca americana L., and ecological factors including understory irradiance, soil stoichiometry and microbial patterns associated with these traits in different exotic plantations of Robinia pseudoacacia L. and Pinus thunbergii Parl. at Mount Lao, Qingdao, China. We found that the traits of P. americana underneath the R. pseudoacacia stand might be situated at the fast side of the trait economic spectrum. The R. pseudoacacia stand appeared to “nurse” P. americana. Furthermore, we intended to explain the nurse effects of R. pseudoacacia stands by examining their ecological factors. First, the R. pseudoacacia stand created understory light attenuation, which matched the sciophilous feature of P. americana. Second, the soil beneath the R. pseudoacacia stand might benefit P. americana more since the soil has greater resource availability. Third, a higher microbial diversity was found in the soil derived from P. americana underneath the R. pseudoacacia stand. A greater abundance of plant pathogens was detected in the soil derived from P. americana in the R. pseudoacacia stand, while more abundant mycorrhizal fungi were detected in the P. thunbergii stand. We speculate that plant pathogens can defend P. americana from aggression from other understory competitors. The mycorrhizal fungi in the P. thunbergii stand might benefit P. americana while simultaneously benefiting other understory plants. Intensive competition from other plants might interfere with P. americana. The potential relationships between plant performance and ecological factors may explain the invasion mechanism of P. americana. The present study provides a novel insight on the facilitative effects of exotic tree plantation on an exotic herb through the modification of soil biota, with implications for the biocontrol of invasive species and forest management and conservation.

Furthermore, we intended to explain the nurse effects of R. pseudoacacia stands by examining their ecological factors.First, the R. pseudoacacia stand created understory light attenuation, which matched the sciophilous feature of P. americana.Second, the soil beneath the R. pseudoacacia stand might benefit P. americana more since the soil has greater resource availability.Third, a higher microbial diversity was found in the soil derived from P. americana underneath the R. pseudoacacia stand.A greater abundance of plant pathogens was detected in the soil derived from P. americana in the R. pseudoacacia stand, while more abundant mycorrhizal fungi were detected in the P. thunbergii stand.We speculate that plant pathogens can defend P. americana from aggression from other understory competitors.The mycorrhizal fungi in the P. thunbergii stand might benefit P. americana while simultaneously benefiting other understory plants.Intensive competition from other plants might interfere with P. americana.The potential relationships between plant performance and ecological factors may explain the invasion mechanism of P. americana.The present study provides a novel insight on the facilitative effects of exotic tree plantation on an exotic herb through the modification of soil biota, with implications for the biocontrol of invasive species and forest management and conservation.

| INTRODUC TI ON
Forests provides benign understory habitats for several shrubs and herbs, protects them from the harm of overheating, promotes their survival and establishment and causes beneficiary "nurse effects" (Cavieres et al., 2005).Therefore, an increasing number of species have invaded forest habitats, especially plantations of exotic trees (Kumar et al., 2020;Lapin et al., 2019).In addition, nurse plants can play a role in the ecological engineering of species by modifying soil conditions and "luring" the invasion of exotic plants.Invasional meltdown and mutualistic interactions among exotic invaders may underlie "nurse effects," which may promote their coinvasion and boost the impacts of invasion on native ecosystems (Simberloff & Von Holle, 1999).For instance, Flory and Bauer (2014) reported that the invasion of Microstegium vimineum enhanced habitat resource availability and facilitated the secondary invasion of Alliaria petiolata in a long-term field experiment.Niu et al. (2007) reported that the soil biota at sites occupied by exotic plants inhibited the growth of native species more than they did at sites occupied by invasive species.Jordan et al. (2008) showed that invasive plants modify the soil microbiota, which promotes invasion directly or via "cross-facilitation" of other invasive species.Zhang et al. (2020) found that exotic plants were more competitive than their native counterparts in soil conditioned by other exotics and that soil microbes drove the invasion success of exotic plants in a multispecies pot experiment.
The facilitative effects of nurse plants can be evaluated by the amelioration of the growth of beneficiary species (Al-Namazi & Bonser, 2020;Garrote et al., 2022;Lozano et al., 2020).Plant life-history traits are representative of plant fitness since they are equivalents of growth, survival and reproduction to a certain extent (Laughlin et al., 2020).Leaf and root functional traits are common indices for the resource use of above-(e.g., light and carbon) and belowground resources (e.g., soil nutrients) (Funk et al., 2016).The leaf trait economic spectrum and the root trait economic spectrum have been proposed for the analysis of correlations between traits and resource use (Fajardo & Siefert, 2018;Weemstra et al., 2016).For instance, plants with a high specific leaf area (SLA) are considered to exhibit rapid growth since plants with a high SLA usually return quickly to their investment in nutrients and leaf mass and thus exhibit a high growth rate (Leishman et al., 2007;Montesinos, 2022).The foliar nutrient concentration (e.g., leaf nitrogen concentration) usually positively correlates with photosynthetic efficiency, as nitrogen is a constituent of chlorophyll and photosynthesis-related enzymes (Taiz & Zeiger, 2010;Wang et al., 2017).In correspondence with leaf functional traits, root functional traits such as the root nutrient reserve may indicate the resource use of belowground nutrients (Weemstra et al., 2016).However, plant root systems are diverse; for example, storage roots function as repositories for nutrient reserves (Vanderschuren & Agusti, 2020).The nutrient concentration of storage roots is associated with asexual reproductive capacity and stress tolerance (Yin et al., 2020).
Afforestation with exotic plantations has been pervasive across natural, seminatural and artificial habitats all over the globe and has caused both beneficiary and harmful impacts to the local ecosystems (Baard et al., 2024;Gómez-González et al., 2024;Reisman-Berman et al., 2019).Mount Lao, which is located in Qingdao city, China, has a vast area of artificial tree plantations (Zhang et al., 2015).Due to the poor site conditions for plant growth (e.g., thin soil layer, granitic body, and soil nutrient deficiency), several tolerable tree species have been introduced for silviculture and revegetation.Two exotic species, R. pseudoacacia (originating from North America) and Pinus thunbergii (originating from Korean Peninsula and Japan), which were introduced in the early 20th century and commonly used for afforestation in Shandong Province, China, dominate plantations and usually form pure stands (Li et al., 2022)., 2016).Since this species has a morphology similar to that of its native congener, also a traditional Chinese medicine, Phytolacca acinosa Roxb., people tend to eat P. americana for medical use by mistake, and the poisonous chemical substances of P. americana induce diarrhea and cause other health problems (Xiao et al., 2022).Due to the sciophilous character of the exotic herb P. americana, the understory of R. pseudoacacia and P. thunbergii stands has been invaded by this species, and several birds assist in the dispersal of this species in the forest ecosystem of Mount Lao (Xiao et al., 2022).Thus, the plantations of R. pseudoacacia and P. thunbergii most likely served as nurses for P. americana.In particular, the Robinia pseudoacacia stand has been favored by several exotic plant species, and an invasive meltdown-the mutual benefit between invaders-was proven between R. pseudoacacia and plant invaders (Medvecká et al., 2018).In addition, using a pot experiment, Chen et al. ( 2019) functional traits, invasion mechanism, light condition, plant-plant interaction, soil microbes, soil stoichiometry, understory invader

T A X O N O M Y C L A S S I F I C A T I O N
Microbial ecology, Microbiomics, Population ecology, Soil ecology found that P. americana performed better in soil collected from R. pseudoacacia plantations than in soil derived from natural forests due to the greater resource availability of R. pseudoacacia plantations.In this study, traits relevant to the growth and reproduction (indicators of invasion) of P. americana in the understory of R. pseudoacacia and P. thunbergii stands were compared in situ.
Additionally, enlightened by previous studies, the soil beneath each sampled plant in each stand type was analyzed for its stoichiometry and microbial pattern (indicators of invasibility).Two hypotheses were proposed as follows: 1.The stand type determines the trait performance relevant to the growth and reproduction of P. americana, and the R. pseudoacacia stand had a stronger nurse effect on P. americana than did the P. thunbergii stand.
2. Ecological factors including understory irradiance, soil stoichiometric and microbial patterns, determine the trait performance of P. americana and are related to its invasion success in R. pseudoacacia stands.
The present study is important for conservation practitioners, forest managers and forestry decision-makers since practical strategies for the management of understory invaders can be proposed based on the current outcomes.
Qingdao is located in the temperate region of China and has a hybrid monsoon climate and marine climate.The average annual temperature is 12.7°C, and the average annual precipitation is 700 ± 100 mm (Guo et al., 2023).The sampled plants were similar in size within the understory of each tree stand.The plant height was recorded.The distance between any two sample plants was greater than 5 m.Five, 6, and 6 P. americana plants (for a total of 17 plants) were sampled from the 3 R. pseudoacacia plots, and 6, 7 and 7 P. americana plants (for a total of 20 plants) were sampled from the 3 P. thunbergii plots.Five leaves at the third node of the branches were randomly harvested and labeled.A piece of storage roots (diameter size: 1-4 cm) -morphologically and physiologically modified underground organs with a major function of nutrient reserve were cut from the belowground parts of each sampled plant and labeled.
The soil underneath the plant (around the roots, 20 cm in depth) was sampled after the sampling of leaves and storage roots.All the sampled materials were immediately placed in an ice box after each sampling and returned to the refrigerator at −20°C for storage in the laboratory.

| Functional trait measurement
High-definition images of the five sampled leaves (600 dpi) were generated using an Epson Perfection v19 scanner with a ruler on the scanning board.The area per leaf was calculated using ImageJ 1.46 (National Institutes of Health, Bethesda, Maryland, USA).Then, each leaf was labeled and oven-dried at 72°C for 48 h.The dry weight per leaf was determined using an analytical balance.The specific leaf area (SLA) was calculated as follows: The sampled storage roots were oven-dried at 72°C for 48 h.
Then, the dry materials of the leaves and storage roots were ground into powder for the analyses of carbon (C) and nitrogen (N) concentrations per leaf mass (leaf C and leaf N) and per root mass (root C and root N) (EuroEA3000 CHNS-O analyzer, Euro Vector, Italy).The leaf carbon/nitrogen ratio (leaf C/N) and root carbon/nitrogen ratio (root C/N) were calculated as follows: The remnant plant materials were oven-dried at 72°C for 72 h.
Then, the biomass of each sampled plant was determined.

| Soil elemental analysis
The soil in Mount Lao (brown soil) is an Eutric Cambisol according to the soil classification system of the United Nations (FAO & IIASA, 2023).The sampled soil was placed in a dry and ventilated  sample was ground into powder and sieved.The soil carbon and nitrogen contents (soil C and soil N) were measured using a EuroEA3000 CHNS-O analyzer (EuroVector, Italy) (Wang et al., 2017).Then, the soil C/N was calculated as the soil C/soil N.

| Extraction of genomic DNA
The CTAB method was used to extract whole-genome DNA from the soil samples.The DNA concentration and purity were monitored on 1% agarose gels.Then, sterilized water was added to dilute the DNA to 1 ng μL −1 .

| PCR product quantification and qualification
The same volumes of 1× loading buffer (containing SYB green) and PCR products were mixed, and electrophoresis was performed on a 2% agarose gel for detection.The PCR products were mixed at equal density ratios.The Qiagen Gel Extraction Kit was subsequently used to purify the mixture of PCR products (Qiagen, Germany).

| Library preparation and sequencing
A TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) was used to generate sequencing libraries.Library quality was assessed using a Qubit@ 2.0 Fluorometer (Thermo Scientific) and an Agilent Bioanalyzer 2100 system.Finally, an Illumina NovaSeq

F I G U R E 1
The location of sampling site for Phytolacca americana in the understory of respective three pure stands of Robinia pseudoacacia and Pinus thunbergii.
platform was used for sequencing the library, and 250 bp paired-end reads were produced.

Data split
The unique barcodes of paired-end reads were assigned to the samples, which were subsequently truncated by removing the barcode and primer sequence.

Chimera removal
The UCHIME algorithm (UCHIME Algorithm, http:// www.drive5. com/ usear ch/ manual/ uchime_ algo.html) was used for comparisons between the tags and the reference database (Silva database).Then, the chimeric sequences were detected and removed.The effective tags were obtained.

OTU production
Uparse software (Uparse v7.0.1001, http:// drive5.com/ uparse/ ) was used for sequence analysis.Sequences with more than 97% similarity were categorized into the same OTUs.A representative sequence of each OTU was screened for further annotation.

Species annotation
The Mothur algorithm was used to annotate taxonomy according to the Silva Database (http:// www.arb-silva.de/ ).

Data normalization
A standard sequence number corresponding to the sample with the least number of sequences was used to normalize the abundance of OTUs.

Alpha diversity
Shannon's index was used for the analysis of species diversity within a sample.The index in our samples were calculated with QIIME (version 1.9.1) and visualized using R software (version 2.15.3).

Beta diversity
Beta diversity (β-diversity), which indicates the complexity of the community composition, was determined through weighted and unweighted UniFrac distances in QIIME (version 1.9.1).

| Growth traits
The biomass, SLA, leaf N, and root N of P. americana were signifi-

| Soil stoichiometry
A significantly greater soil N and a significantly lower soil C/N were shown in the soil derived from P. americana underneath the R. pseudoacacia stand than in that derived from the P. thunbergii stand (p < .1)(Figure 3a,b).

| Diversity
Generally, the observed species and Shannon's index of bacteria and fungi were significantly greater for the soil derived from P. americana underneath the R. pseudoacacia stand than for that derived from the soil underneath the P. thunbergii stand (p < .1)(Figure 4a-d).

| Generic composition
Considering the soil bacteria, P. americana accumulated a greater relative abundance of Streptomyces but a lower relative abundance of Acidipila-Silvibacterium, Bradyrhizobium, Corynebacterium, Bryobacter, and Pseudomonas underneath the R. pseudoacacia stand than underneath the P. thunbergii stand (Figure 5).
Considering soil fungi, the soil derived from P. americana in the understory of the R. pseudoacacia stand had a greater relative abundance of Fusarium and Auxarthron species than that in the understory of the P. thunbergii stand did, while Russula, Umbelopsis, and Geminibasidium species were more abundant underneath the P.

| Correlations between plant traits and ecological factors
Positive correlations were shown between biomass and bacterial observed species, biomass and bacterial Shannon's index, SLA and and SLA has been observed (Leishman et al., 2007).Considering the probable positive correlation between SLA and relative growth rate, a greater leaf N and a lower leaf C/N usually indicate a faster plant growth (Leishman et al., 2007;Montesinos, 2022).Root form, including root morphology, architecture and chemical parameters, is linked with root function (Iversen, 2014).On a geometric basis, roots with a herringbone branching structure have been shown to have greater nutrient absorption capacity than those with a dichotomous branching structure (Fitter et al., 1991).Thicker roots with longer lifespans and higher mycorrhizal colonization rates usually indicate higher construction costs and lower nutrient assimilation rates, while a higher root branching intensity is usually equivalent to greater proliferation in patches with abundant nutrients (Kong et al., 2014).Root morphological traits such as specific root length (SRL) and specific root area (SRA) are proxies for nutrient assimilation in plants (Weemstra et al., 2016).We also observed positive correlations between biomass and bacterial observed species, biomass and bacterial Shannon's index, SLA and fungal Shannon's index, leaf N and fungal Shannon's index.
Considering the soil bacteria, P. americana "gathered" a greater abundance of Streptomyces in the understory of R. pseudoacacia stand.
Several Streptomyces species can symbiose with plants and secrete antibiotics which may probably protect P. americana from infection and indirectly promote its growth (Seipke et al., 2012).However, some Streptomyces species, such as S. scabies, S. turgidiscabies and S. acidiscabies, can cause potato scab disease in tuber plants, and P.
americana, which has a large root tuber, may be a sufferer (Bignell et al., 2010).As the ratio of "beneficial" to "harmful" Streptomyces Phytolacca americana L. (originating from North America) has been officially listed as an exotic invasive species by the Ministry of Ecology and Environment of the People's Republic of China since this species can establish pervasive monostands, rapidly deplete soil nutrients, exclude native species by resource competition and allelopathy and threaten local biodiversity (Ministry of Ecology and Environment of the People's Republic of China & Chinese Academy of Sciences room and naturally weathered to a constant weight.Then, each soil SLA = dry weight per leaf ∕ leaf area per leaf leaf C ∕ N = leaf C ∕ leaf N root C ∕ N = root C ∕ root N TA B L E 1 The characteristics for the respective three sampling 20 m × 30 m plots of Robinia pseudoacacia and Pinus thunbergii pure stands. Specific primers (e.g., 16S V4: 515F806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R, etc.) were used to amplify the 16S rRNA/18SrRNA/ ITS genes of distinct regions (16S V4/16S V3/16S V3-V4/16S V4-V5, 18S V4/18S V9, ITS1/ITS2, and Arc V4) with barcodes.All PCRs were performed with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 2 μM forward and reverse primers, and approximately 10 ng of template DNA.Thermal cycling included 1 min of initial denaturation at 98°C, 30 cycles of denaturation at 98°C for 10 s, 30 s of annealing at 50°C, 30 s of elongation at 72°C and a 5 min extension at 72°C.
greater underneath the R. pseudoacacia stand than underneath the P. thunbergii stand (p < .1)(Figure 2a-c,e).The leaf C/N of P. americana were significantly lower underneath the R. pseudoacacia stand than underneath the P. thunbergii stand (p < .1),while no significant difference of root C/N was shown for P. americana underneath different stands (p > .1)(Figure 2d,f).

F I G U R E 2 |
Comparison in the growth traits including (a) biomass, (b) SLA, (c) leaf N, (d) leaf C/N, (e) root N and (f) root C/N of Phytolacca americana underneath different stand types of Robinia pseudoacacia and Pinus thunbergii.Values are mean ± standard error.fungal observed species, leaf N and fungal Shannon's index, leaf C/N and soil C/N, root N and soil N (Figure 7).Negative correlations were displayed between biomass and understory irradiance, biomass and soil C/N, SLA and soil C/N, leaf N and soil C/N, leaf C/N and fungalShannon's index, root C/N and soil N (Figure7).Advantages in terms of growth traits contributed to the thriving of invasive P. americana in the understory of R. pseudoacacia stands An overwhelming advantage in growth was shown for P. americana in the understory of R. pseudoacacia compared to that in the understory of P. thunbergii.First, the biomass was approximately 36 times greater in the understory of R. pseudoacacia than in the understory of P. thunbergii.Since biomass are usually positively correlated with fecundity, biomass has been considered to represent fitness(Younginger et al., 2017).Second, P. americana in the understory of R. pseudoacacia had a greater SLA.A greater SLA usually predicts a greater relative growth rate(Leishman et al., 2007;Montesinos, 2022).Third, considering tissue stoichiometry, P. americana underneath the R. pseudoacacia stand had greater values in leaf N and root N but lower leaf C/N.The leaf trait economic spectrum indicates a positive correlation between leaf N and SLA, and a negative correlation between leaf C/N Root type is another deterministic factor in root function.Considering the chemical properties of roots, root N has potential positive correlations with nutrient assimilation for absorbing roots and with nutrient storage for storage roots separately, while nutrient assimilation determines the plant growth and root nutrient storage affects the clonal reproduction(Weemstra et al., 2016;Yang et al., 2020).Therefore, considering the mixing of both root types of P. americana, P. americana in the understory of R. pseudoacacia may have faster growth and regrowth.In summary, compared with the P. thunbergii stand, the R. pseudoacacia stand can facilitate the invasion success of P. americana as a nurse plant.An invasional meltdown between R. pseudoacacia and P. americana may occur, followed by a probable rigid vertical community structure of trees and herbs, which may efficiently resist the intrusion of other species and accelerate the impact of coinvasion on local ecosystems(Simberloff & Von Holle, 1999).4.2 | Understory irradiance, soil stoichiometry, and microbial pattern shaped the performance of P. americana in different exotic standsUnderstory irradiance, soil stoichiometry and soil microbial patterns determine the invasion success of P. americana.First, a negative correlation between biomass and understory irradiance was shown, which indicates the sciophilous characteristic of P. americana(Xiao et al., 2022).The R. pseudoacacia stand can cause greater light degradation than the P. thunbergii stand in its understory and promotes the growth of P. americana.Second, in the perspective of soil stoichiometry, a higher fertility was detected in the soil in the understory of R. pseudoacacia, which totally synchronizes with the fast trait economics of P. americana(Chen et al., 2019).Redundancy analysis (RDA) revealed negative correlations between biomass and soil C/N, SLA and soil C/N, leaf N and soil C/N, root C/N and soil N; however, positive correlations between leaf C/N and soil C/N, root F I G U R E 3 Comparison in the soil stoichiometry including: (a) soil N and (b) soil C/N derived from Phytolacca americana underneath different stand types of Robinia pseudoacacia and Pinus thunbergii.Values are mean ± standard error.N and soil N were observed.Hence, greater habitat nutrient (e.g.nitrogen) availability tends to "shape" a faster trait economics for P. americana and results in faster growth of the exotic invader(Fajardo & Siefert, 2018;Montesinos, 2022;Weemstra et al., 2016).This observation is consistent with the fluctuating resource hypothesis on invasion mechanisms, as an increase in environmental resources provides surplus niches for invaders to colonize(Liu et al., 2020;Pearson et al., 2018;Wohlgemuth et al., 2022).Third, the observed species and Shannon's index of both bacteria and fungi increased in the soil derived from the understory of the R. pseudoacacia stand, which indicates the "prosperity" of the soil microbes underneath the R. pseudoacacia stand.Previous studies have proposed a F I G U R E 4 Comparison in the soil microbial diversity including: (a) bacterial observed species, (b) bacterial Shannon's index, (c) fungal observed species and (d) fungal Shannon's index derived from Phytolacca americana underneath different stand types of Robinia pseudoacacia and Pinus thunbergii.Values are mean ± standard error.F I G U R E 5 Comparison in the relative abundance of top 10 soil bacterial genera derived from Phytolacca americana underneath different stand types of Robinia pseudoacacia and Pinus thunbergii.probablecorrelation between soil microbial diversity especially the abundance of beneficial microbes, and invasion success(Dawson & Schrama, 2016;Meng et al., 2024).Plants and soil microbes generally mutually affect each other, as strong plant growth usually induces the proliferation of soil microbes and vice versa(Thakur et al., 2021).
species was not quantified in the present study, the effects of Streptomyces species on the performance of P. americana were uncertain.Simultaneously, greater abundances of Bradyrhizobium and Bryobacter existed in the soil derived from P. americana underneath the P. thunbergii stand.The Bradyrhizobium species can symbiose with legume and nonleguminous plants and assist in the assimilation of soil nutrients, such as nitrogen fixation (de Alencar MenezesJúnior et al., 2019).Bryobacter species aid in the decomposition of lignin and cellulose, promote the carbon cycling and are considered to benefit plant growth(Yu et al., 2016).However, since the soil fertility was lower and the diversity of neighboring species of P. americana was greater (based on our field observations) underneath F I G U R E 6 Comparison in the relative abundance of top 10 soil fungal genera derived from Phytolacca americana underneath different stand types of Robinia pseudoacacia and Pinus thunbergii.F I G U R E 7 Redundancy analysis (RDA) of the correlation between growth traits of Phytolacca americana including biomass, SLA, leaf N, leaf C/N, root N, and root C/N, and environmental variables including understory irradiance (irradiance), soil N, soil C/N, observed species B (bacterial observed species), Shannon B (bacterial Shannon's index), observed species F (fungal observed species) and Shannon F (fungal Shannon's index).